Preterm birth is the leading cause of infant mortality, and has been targeted for funding by the NICHD Pregnancy and Perinatalogy Branch. Two large independent randomized clinical trials showed that administration of 17a-hydroxyprogesterone caproate (17P) or progesterone (P4) during the 2nd trimester to women at risk of spontaneous preterm birth resulted in a significant reduction in the incidence of preterm labor. Progestins induce myometrial quiescence, likely due in part to alterations in P4 receptor-mediated gene expression in myometrial cells. However, the effects of P4 signaling on the gene expression profile of myometrial cells are largely unknown. The objective of this project is to define progestin-regulated gene networks in human myometrial cells, to provide a deeper understanding of progestin control of myometrial contractility. Unraveling these intricate pathways is essential to understanding biological mechanisms that control myometrial function and could lead to the development of improved and/or novel tocolytics. A standard approach to profiling transcriptomes has been to use cultured cells. However, myometrial cells in primary culture rapidly lose P4 (and estrogen) receptors. Both estrogen and P4 signaling are essential in reproductive processes, and estrogens up-regulate both estrogen and P4 receptor gene expression in all mammalian species. We will use recombinant methods to reengineer immortalized myometrial cell lines developed in our laboratory so that they conditionally express estrogen receptors. Endogenous P4 receptors will then be induced by the administration of estrogen. Using GeneChip microarray analysis, the cells will be used to characterize the human myometrial cell transcriptome before and after 17P or P4 administration. Changes in expression of transcripts that show a likely relationship to uterine contractile activity will be confirmed by real time PCR. PCR will also be use to verify gene repression, using intron/exon oligonucleotides to probe newly synthesized transcripts. The recognition of progestin target genes in human myometrial cells, coupled with the use of knowledge-based analysis tools to model and understand complex biological systems, will allow us to begin to identify biological networks involved in progestin maintenance of uterine quiescence, and serve to guide more elaborative biochemical studies on the interplay between progestin regulated processes. Two large, independent, randomized clinical trials showed that administration of 17a-hydroxyprogesterone caproate or progesterone during the second trimester to women at risk of spontaneous preterm birth resulted in a significant reduction in the incidence of preterm labor. The objective of this project is to define progestin-regulated gene networks in human myometrial cells, to provide a deeper understanding of progestin control of myometrial contractility. Unraveling these intricate pathways is essential to understanding biological mechanisms that control myometrial function and could lead to the development of improved and/or novel tocolytics. [unreadable] [unreadable] [unreadable]